Reagent for detecting and monitoring viral infections

ABSTRACT

The invention concerns a reagent for diagnosing an infection caused by a virus, characterized in that it comprises essentially a mixture consisting of (1) an immunodominant fragment of a protein of said virus comprising not more than 60 aminoacids, preferably between 20 and 30 aminoacids and (2) a mixture (called mixotope) of convergent combining peptides, derived from said immunodominant fragment, which peptides are obtained by total or partial artificial degeneration of said immunodominant fragment by systematic or partial replacement of each aminoacid by another according to an appropriate substitution matrix. The invention concerns a reagent for detecting and monitoring infections caused by the Epstein-Barr virus of EBV, which is, in particular, the causal agent of infectious mononucleose and its applications for detecting an EBV infection at any stage of the infection (primo-infection, healthy carriers and induced tumors). Said diagnosis reagent comprises essentially a mixture consisting of (1) a C-terminal fragment of the protein VCAp18 SEQ ID n o  1 of the Epstein-Barr virus (EBV) comprising not more than 60 aminoacids, preferably between 20 and 30 aminoacids, and (2) a mixture of convergent combining peptides, derived from said C-terminal fragment.

The present invention relates to a reagent for detecting and monitoringviral infections, such as those caused by the Epstein-Barr virus or EBV,which is in particular the causative agent of infectious mononucleosis,by the hepatitis C virus (HCV) or by the human immunodeficiency virus(HIV) and to its applications for the detection of a viral infection, inparticular of an EBV infection, at any stage of the infection (primaryinfection, healthy carriers and induced tumours).

EBV is a herpesvirus which preferably infects the B lymphocytes and theepithelial cells.

This lymphocryptic virus escapes immunological surveillance. In general,the virus is carried by healthy carriers, with no special symptoms;however, under immunodeficient conditions, such as AIDS or chemotherapyfollowing a transplant, as well as in endemic regions, such as in Africaor in Asia, the oncogenic potential of EBV is freed and leads to theemergence of various tumours, such as African Burkitt's lymphoma,undifferentiated carcinoma of the nasopharynx, certain lymphomas andHodgkin's disease.

At present the serodiagnosis of EBV is carried out either by thePaul-Bunnell reaction (detection of heterophilic antibodies), a simpleand rapid but nonspecific method (LINDERHOLM M. et al., J. Clin.Microbiol., 1994, 32, 1, 259-261), by immunofluorescence (IF) testswhich are specific for each class of antigens (VCA, EA and EBNA).

The development of a simpler and less expensive diagnostic test using animmunoenzymatic method such as the ELISA method is in this contextparticularly desirable and several tests have thus been proposed (M.GORGIEVSKY-HRISOHO et al., J. Clin. Microbiol., 1990, 28, 2305-2311; JM. MIDDELDORP et al., J. Virol. Methods, 1988, 21, 133-159).

The immunoenzymatic methods proposed in the prior art exhibit, for themajority, the major disadvantage of lacking detection sensitivity (M.GORGIEVSKY-HRISOHO et al., cited above; J M. MIDDELDORP et al., citedabove). To solve this problem of lack of sensitivity, some authors haveproposed using various antigens, in combination; such combinationsincrease the sensitivity of the test in which these combinations areused, but not the specificity. The combinations of antigens which havebeen proposed in the prior art comprise, for example, the antigen p47-52(major antigen EA-D, encoded by the open reading frame called BMRF1,SWISS-PROT accession No. P03191), the antigen p38 (encoded by the openreading frame called BALF2, SWISS-PROT accession No. P03227) and theantigen gp125 or gp110 (encoded by the open reading frame called BALF4,SWISS-PROT accession No. P03188) (W. M. J. Van GRUNSVEN et al., J.Virol., 1993, 67, 3908-3916).

More recently, it has been shown that an 18 kDa capsid antigen (VCAp18),encoded by the open reading frame BFRF3 (SWISS-PROT accession No.P14348), is recognized by healthy carriers of EBV in immunoblot analysisand appears not to exhibit homologous sequences with the other humanherpesviruses, unlike the major proteins, such as VCAp40 (sequenceencoded by the open reading frame called BdRF1, SWISS-PROT accession No.P03219) and gp125/110 (R. BAER et al., Nature, 1984, 310, 207-211; M. S.CHEE et al., Curr. Topics Microbiol. Immunol., 1990, 154, 125-170; A. J.DAVIDSON, J. Gen. Virol., 1986, 67, 1759-1816). The map of the antigenicdomains of the antigen VCAp18 has been established and the majorantigenic domain has been localized in the C-terminal region of theprotein (W. M. J. Van GRUNSVEN et al., J. Infect. Dis., 1994, 170,13-18).

However, ELISA tests carried out with this capsid antigen VCAp18 havethe disadvantage:

of not exhibiting sufficient sensitivity and specificity to detect allthe anti-VCA Ig's produced (false-positives and/or false-negatives); inparticular, as regards the false-negatives, they are essentially due tothe small size of the synthetic peptide VCAp18/SEQ ID No. 2 (24 aminoacids, SEQ ID No. 2 of the sequence listing), which may consequently notbe recognized by all the VCA-positive individuals and/or because of theheterogeneity of the reactivity of the antibodies towards the syntheticpeptide, compared with the same peptide, in its natural environment,

of not allowing differential diagnosis of the various stages of the EBVinfection and/or of the pathologies induced by EBV, depending on theisotype profile of the Ig's produced (IgG, IgA and IgM), despite the useof the C-terminal fragment of the VCAp18 protein (SEQ ID No. 1 of thesequence listing), as reagent.

A similar situation is encountered with other viruses, such as HCV orHIV; indeed, in general, the use of one or more immunodominant fragmentsdoes not exhibit sufficient sensitivity to avoid false-negatives.

Accordingly, the Applicant company set itself the objective of providinga new reagent for the detection of viral infections, capable of beingused in immunoenzymatic tests, which is both specific and sensitive andwhich makes it possible to obtain a gain in sensitivity of at least 15to 30% compared with the prior art reagents.

Accordingly, the Applicant company also set itself the objective ofproviding a new reagent for the detection of EBV infections, capable ofbeing used in immunoenzymatic tests, which is both specific andsensitive and which allows differential diagnosis of the stage of theinfection and/or of the pathology, depending on the prevalent isotype;indeed, the presence of human anti-VCA IgMs is essentially the sign of aprimary infection, the presence of human anti-VCA IgGs is essentiallythe sign of a past infection (healthy carriers, generally), whereas thepresence of human anti-VCA IgAs suggests the emergence of a tumour. Sucha reagent is more suitable for the requirements of practical use thanthe prior art reagents in the context of immunoenzymatic tests, inparticular of the ELISA type.

The subject of the present invention is a reagent for the diagnosis ofan infection caused by a virus, characterized in that it comprisesessentially a mixture consisting of (1) an immunodominant fragment of aprotein of the said virus comprising at most 60 amino acids, preferablybetween 20 and 30 amino acids and (2) a mixture (called mixotope) ofconvergent combinatory peptides derived from the said immunodominantfragment, which peptides are obtained by complete or partial artificialdegeneration of the said immunodominant fragment by systematic orpartial replacement of each amino acid with another according to asuitable replaceability matrix.

For the purposes of the invention, mixotope is understood to mean themixture of all the combinatory peptides obtained from the selectedimmunodominant fragment by artificial or constructed degeneration; theyare preferably obtained during a single synthesis and represent thepeptide antigen and its variability in its antibody populationrecognition function; various mixotopes may be obtained from the samepeptide; the factors which are involved in the constitution of amixotope are:

on the one hand, the percentage degeneration of the nativeimmunodominant fragment selected (total or partial degeneration); theconserved amino acids (isolated or forming a sequence), in the case of apartial degeneration, are preferably, as regards EBV, those which areinvolved in the structuring of the VCAp18 protein and

on the other hand, the mode of selection of the substitution of theamino acids of the said native immunodominant fragment; for eachposition of the sequence of the native immunodominant fragment chosen,the amino acid substitution is selected on the basis of the replacementmatrix established by H. M. GEYSEN et al., (J. Mol. Recog., 1988, 1,32-41), or modified, as illustrated in FIG. 16, taking into account thetolerance of the antibody recognition, depending on the amino acidsubstitution in the linear epitopes: for a given position, the aminoacids exhibiting the highest percentage of “replaceability” arepreferably chosen. However, it is preferable to take into account theconformation of the natural epitopes, before degeneration.

The mixotope for the purposes of the present invention, consisting ofconvergent combinatory peptides derived from a native immunodominantfragment, therefore represents an artificial and non-naturaldegeneration of the native structure by the systematic or partialreplacement of each amino acid with another derived from the GEYSENreplaceability matrix or from the matrix according to FIG. 16.

Also for the purposes of the invention, constructed replaceabilitymatrix is understood to mean a matrix which does not reproduce thenatural variations or the variations frequently observed in evolution;the GEYSEN replaceability matrix or the matrix according to FIG. 16 areparticularly suitable.

Surprisingly, the reagents according to the invention make it possibleto obtain reliable, reproducible, very sensitive and very specificresults since the combinations according to the invention exhibit asynergistic effect in the detection of the antibodies induced byviruses; in particular, a gain in sensitivity of 15 to 30% is generallyobtained regardless of the virus (HIV, HCV, EBV, for example).

The subject of the present invention is also a diagnostic reagentaccording to the invention, characterized in that, for the diagnosis ofEBV infections, it comprises essentially a mixture consisting of (1) aC-terminal fragment of the 18 kDa viral capsid antigen (protein VCAp18,SEQ ID No. 1) of the Epstein-Barr virus (EBV) comprising at most 60amino acids, preferably between 20 and 30 amino acids and (2) a mixture(called mixotope) of convergent combinatory peptides derived from thesaid C-terminal fragment or peptide VCAp18.

According to an advantageous embodiment of the reagent according to theinvention, the said C-terminal fragment of the VCAp18 protein isselected from the group consisting of the peptides VCAp18/SEQ ID No. 2,VCAp18/SEQ ID No. 3, VCAp18/SEQ ID No. 4 and VCAp18/SEQ ID No. 5.

Unexpectedly, the combination of a C-terminal fragment of the VCAp18protein with a mixotope, derived from the said fragment, and regardlessof the configuration of the C-terminal fragment chosen, significantlyenhances the sensitivity and specificity of the immunoenzymaticserodiagnosis of EBV; indeed, if an ELISA test using a reagent accordingto the invention as defined above is compared with an ELISA test usingonly a peptide VCAp18, a 100% sensitivity and a 100% specificity areeffectively obtained with the reagent according to the invention becauseof the binding of all the anti-VCA Ig's (G, A and M) to the saidreagent.

Surprisingly, the reagents according to the invention make it possibleto obtain reliable, reproducible, very sensitive and very specificresults because the combinations according to the invention exhibit asynergistic effect in the detection of all the human anti-VCA Ig'sproduced.

The mixture of degenerate peptides which is artificially produced duringthe same synthesis is combined with the native peptide. Such acombination makes it possible, unexpectedly, to increase the reactivityof the mixture of antigens which is produced with respect to theantibodies naturally induced by the virus.

Under these conditions, it is possible to obtain sensitivities of theorder of 100% and 97%, respectively, in the populations of post-infectedand primary-infected subjects, while preserving 100% specificity in allthe tests carried out.

For example:

for the peptide VCAp18/SEQ ID No. 2, it is thus possible to obtain thefollowing three mixotopes:

the mixotope corresponding to a degeneration of the entire sequence ofthe peptide VCAp18/SEQ ID No. 2, called hereinafter mixotope MIXO,

the mixotope in which the proline residues of the peptide VCAp18/SEQ IDNo. 2 are preferentially conserved, the latter being involved inparticular in the conformation; the said mixotope, called hereinaftermixotope MIXO(P), corresponds to a partial degeneration of the sequenceof the peptide VCAp18/SEQ ID No. 2 and

the mixotope in which both the proline residues and the sequence Gly SerGly Gly Gly Gly (SEQ ID No. 6) of the peptide VCAp18/SEQ ID No. 2 arepreferentially conserved; the mixotope, called hereinafter mixotopeMIXO(P,G), corresponds to a partial degeneration of the sequence of thepeptide VCAp18/SEQ ID No. 2,

for the peptide VCAp18/SEQ ID No. 3, it is thus possible to obtain thefollowing mixotopes:

the mixotope corresponding to a degeneration of the entire sequence ofthe peptide VCAp18/SEQ ID No. 3,

the mixotope in which the proline residue and/or the serine residues ofthe repetitive sequence of the peptide VCAp18/SEQ ID No. 3 arepreferentially conserved; the said mixotope corresponds to a partialdegeneration of the sequence of the peptide VCAp18/SEQ ID No. 3,

the mixotope in which the proline residue and the alanine residues ofthe repetitive sequence of the peptide VCAp18/SEQ ID No. 3 arepreferentially conserved; the said mixotope corresponds to a partialdegeneration of the sequence of the peptide VCAp18/SEQ ID No. 3 and

the mixotope in which both the proline residue, the serine residues andthe alanine residues of the peptide VCAp18/SEQ ID No. 3 arepreferentially conserved,

for the peptide VCAp18/SEQ ID No. 4, it is thus possible to obtain thefollowing mixotopes:

the mixotope corresponding to a degeneration of the entire sequence ofthe peptide VCAp18/SEQ ID No. 4,

the mixotope in which the serine, proline and/or alanine residues and/orthe sequence Gly Ser Gly Gly Gly Gly (SEQ ID No. 6) and/or the sequenceAla Ala Ala Ser Ala Ala Ala Ala (SEQ ID No. 7) of the peptide VCAp18/SEQID No. 4 are preferentially conserved; the said mixotope corresponds toa partial degeneration of the sequence of the peptide VCAp18/SEQ ID No.4,

for the peptide VCAp18/SEQ ID No. 5, it is thus possible to obtain thefollowing mixotopes:

the mixotope corresponding to a degeneration of the entire sequence ofthe peptide VCAp18/SEQ ID No. 5,

the mixotope in which the serine and/or alanine residues of the peptideVCAp18/SEQ ID No. 5 are preferentially conserved; the said mixotopecorresponds to a partial degeneration of the sequence of the peptideVCAp18/SEQ ID No. 5,

the mixotope in which both the serine residues and the sequence Ala AlaAla Ser Ala Ala Ala Ala (SEQ ID No. 7) of the peptide VCAp18/SEQ ID No.5 are preferentially conserved; the said mixotope corresponds to apartial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 5.

The different mixotopes corresponding to a partial degeneration of thechosen peptide VCAp18 conserve a native sequence which is preferablyinvolved in the structuring of the VCAp18 protein.

According to another advantageous embodiment of the reagent according tothe invention, it is attached to a solid support, preferably microtitreplates.

According to another advantageous embodiment of the reagent according tothe invention, the C-terminal peptide:mixotope ratio in the mixture isbetween 1:10 and 1:100.

The subject of the present invention is also a method for the diagnosisof a viral infection, by an immunoenzymatic method, characterized inthat it uses a diagnostic reagent according to the invention.

In particular, as regards EBV infections, the subject of the presentinvention is also a method of diagnosis by an immunoenzymatic method,characterized in that it comprises:

bringing a serum to be analysed into contact with a reagent as definedabove,

the addition of anti-human Ig antibodies coupled to an enzyme, and

the qualitative and/or quantitative revealing of the anti-VCA antibodieswhich may be present in the serum to be analysed by addition of theenzyme substrate.

According to an advantageous embodiment of the said method, itcomprises:

the attachment of a reagent according to the invention onto a support,such as a microtitre plate,

the addition of the serum to be analysed,

the detection of the attachment of the anti-VCA antibodies present inthe said serum by addition of anti-human Ig (G-A-M) antibodies coupledto an enzyme, and

the qualitative and/or quantitative revealing in a spectrophotometer byaddition of the enzyme substrate.

The subject of the present invention is also a method for thesurveillance and differential detection of the stages of an EBVinfection by an immunoenzymatic method, characterized in that itcomprises:

the attachment of a reagent according to the invention onto a support,such as a microtitre plate,

the addition of the serum to be analysed,

the detection of the attachment of the anti-VCA antibodies, which may bepresent in the said serum, by the addition of anti-human Ig antibodiescoupled to an enzyme, which antibodies are selected from the groupconsisting of the anti-human IgG antibodies, the anti-human IgMantibodies and the anti-human IgA antibodies, and

the qualitative or quantitative revealing in a spectrophotometer by theaddition of the enzyme substrate.

The primary infection induces the formation of antibodies directedagainst the different antigens of EBV; they are in particular theantibodies directed against the viral capsid antigen (VCA), theantibodies directed against the nuclear antigen (EBNA), the antibodiesdirected against the early antigen (EA) and the antibodies directedagainst the membrane antigen (MA).

These different antibodies do not appear at the same stage of theinfection; in particular, the anti-VCA Ig's are produced very early andremain present during the entire life of the host. This means that thedetection of the anti-VCA IgMs and IgGs in the human serum reinforcesthe diagnostic value (particularly advantageous), in order to establisha primary infection with EBV.

Depending on the isotype of the anti-VCA Ig's which is detected in theserum to be analysed, it is possible to distinguish the healthy carriersof the virus (prevalence of the IgGs), the primary infections(prevalence of the IgMs) or the emergence of a tumour, in particularcarcinoma of the nasopharynx (prevalence of the IgAs).

Surprisingly, the reagent according to the invention effectively makesit possible to detect the anti-VCA IgAs.

The subject of the present invention is also a method for the earlydetection of the cancer of the nasopharynx by an immunoenzymatic method,characterized in that it comprises:

the attachment of a reagent according to the invention onto a support,such as a microtitre plate,

the addition of the serum to be analysed,

the detection of the attachment of the anti-VCA antibodies, which may bepresent in the said serum, by the addition of anti-human IgA antibodiescoupled to an enzyme, and

the qualitative or quantitative revealing in a spectrophotometer by theaddition of the enzyme substrate.

The subject of the present invention is, in addition, a kit or box forthe diagnosis of a viral infection, characterized in that it comprisesat least one diagnostic reagent according to the invention.

In addition to the preceding features, the invention further comprisesother features which will emerge from the following description whichrefers to exemplary embodiments of the method which is the subject ofthe present invention as well as to the appended drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the reactivity of the Ig-G-A-Ms of 46 EBV-positivesera () and 28 EBV-negative sera () characterized by immunofluorescencewith the peptide SEQ ID No. 2 attached onto a solid support in an amountof 0.1 μg/well (FIGS. 1A and 1B) or in an amount of 1 μg/well (FIG. 1C).The horizontal line represents the cut-off value, corresponding to themean obtained with the negative (EBV-negative) sera+3 standarddeviations (SD). FIG. 1B illustrates more particularly the comparisonbetween the anti-VCA IgG titres obtained by an immunofluorescence (IF)test with the absorbance value obtained (A492) with an ELISA test usingthe peptide SEQ ID No. 2. In FIG. 1B, the symbol represents theEBV-positive SEQ ID No. 2-negative sera. FIGS. 1A and 1C comprise on thex-axis the number of sera and on the y-axis the absorbance at 492 nm,whereas FIG. 1B comprises on the x-axis the IF titres and on the y-axisthe absorbance values obtained in ELISA.

FIG. 2 represents the effect of the mixotopes MIXO, MIXO(P) andMIXO(P,G) on the reactivity of the IgG-A-Ms of the EBV-positive sera ()and the EBV-negative sera () at two different concentrations: 0.1μg/well (FIGS. 2A, 2C and 2E) or at 10 μg/well (FIGS. 2B, 2D, 2F), in anELISA test. The horizontal line represents the cut-off valuecorresponding to the mean of the control sera+3 SD. These differentfigures comprise on the x-axis the number of sera and on the y-axis theabsorbance at 492 nm.

FIG. 3 illustrates the comparison of the behaviours of EBV-positive serawith respect to the peptide VCAp18/SEQ ID No. 2 (FIG. 3A) and thedifferent mixotopes: MIXO (FIG. 3B), MIXO(P) (FIG. 3C), MIXO(P,G) (FIG.3B); the false-negatives obtained with the peptide VCAp18/SEQ ID No. 2(FIG. 3A, ) define the cut-off value. The data in these FIG. 3 representthe absorbance values obtained by ELISA for all the EBV-positive sera,as a function of their IFA titre. The peptide VCAp18/SEQ ID No. 2 andthe mixotopes are used in solid phase respectively at the concentrationsof 0.1 and 10 μg/well.

FIG. 4 illustrates the effect of the combination peptide VCAp18/SEQ IDNo. 2+mixotopes: MIXO (FIG. 4A), MIXO(P) (FIG. 4B) or MIXO(P,G) (FIG.4C), on the IgG-A-M reactivity of the EBV-positive () and EBV-negative() sera in ELISA tests. Each microtitre plate well is sequentiallycoated with 0.1 μg of peptide VCAp18/SEQ ID No. 2 and with 10 μg of oneof the abovementioned mixotopes and brought into contact with the sera.

FIGS. 5A and 5B illustrate the variation of the human serologicalresponses of the peptide VCAp18/SEQ ID No. 2 () and of theabovementioned three mixotopes (MIXO () MIXO(P) () and MIXO(P,G) () attwo different coating concentrations; FIG. 5C illustrates the variationof the serological responses of the peptide VCAp18/SEQ ID No. 2 () andof the abovementioned three mixotopes in combination with the peptideVCAp18/SEQ ID No. 2; VCAp18/SEQ ID No. 2+MIXO (), VCAp18/SEQ ID No.2+MIXO(P) () and VCAp18/SEQ ID No. 2+(P,G) (). For each experiment, thesera having an absorbance at 492 nm of less than 1.3 with the peptideVCAp18/SEQ ID No. 2 are selected and the vertical line delimits theboundary between the EBV-positive and EBV-negative sera.

FIG. 6 illustrates the inhibitions of the binding of the antibodies ofdifferent EBV-positive sera (serum 298 6967, serum 298 7943, serum 2990723 and serum 299 1372) to a solid phase containing the peptideVCAp18/SEQ ID No. 2 (), MIXO (), MIXO(P) () or MIXO(P,G) () byincreasing the concentrations of peptide VCAp18/SEQ ID No. 2. Thisfigure comprises on the x-axis the concentration of peptide VCAp18/SEQID No. 2 and on the y-axis the A/Ao ratio.

FIG. 7 represents the Klotz plots illustrating the binding of theantibody of different EBV-positive sera (serum 298 6967, serum 298 7943,serum 299 0723 and serum 299 1372) to solid phases containing thepeptide VCAp18/SEQ ID No. 2 (),MIXO (), MIXO(P) () or MIXO(P,G) ().

FIG. 8 illustrates the IgG-A-M reactivity (FIG. 8A) and the separatereactivities of the different G, A and M isotypes (FIG. 8B) ofEBV-positive sera (subjects who have previously had an infection, ) andof EBV-negative sera (), with respect to the reagent according to theinvention VCAp18/SEQ ID No. 2+MIXO(P,G).

FIG. 9 illustrates the reactivities of the IgM isotype with respect tothe reagent according to the invention VCAp18/SEQ ID No. 2+MIXO(P,G) ofEBV-positive sera (previous infection, ) and of negative sera (), whichhave not been treated (FIG. 9A), or which have been treated withanti-IgG antibodies (FIG. 9B) or treated by preadsorption of the IgGs(FIG. 9C).

FIG. 10 illustrates the comparison of the IgG-A-M (FIGS. 10A and 10B)and IgG (FIGS. 10C and 10D) reactivities of human sera from patients whohave previously had an infection, with respect to the reagent accordingto the invention VCAp18/SEQ ID No. 2+MIXO(P,G) with an IFA test for thedetection of the anti-VCA IgGs and a Behring ELISA test for thedetection of the anti-(VCA-EBNA-EA) IgGs. The data represent theabsorbance values obtained in ELISA for all the EBV-positive sera, as afunction of their IFA titre or the absorbance values obtained with theBehring ELISA test.

FIG. 11 illustrates the IgG-A-M reactivity (FIG. 11A) and the separatereactivities of the different G, A and M isotypes (FIG. 11B) ofEBV-positive sera (primary infection, ) and of EBV-negative sera (),with respect to the reagent according to the invention VCAp18/SEQ ID No.2+MIXO(P,G).

FIG. 12 illustrates the reactivities of the IgM isotype with respect tothe reagent according to the invention VCAp18/SEQ ID No. 2+MIXO(P,G) ofEBV-positive sera (primary infection, ) and of EBV-negative sera (),which are not treated (FIG. 12A), or which are treated with anti-IgGantibodies (FIG. 12B) or treated by preadsorption of the IgGs (FIG.12C).

FIG. 13 illustrates the comparison of the IgM reactivity of sera fromprimary-infected patients with respect to the reagent according to theinvention VCAp18/SEQ ID No. 2+MIXO(P,G) with a Behring ELISA test forthe detection of the anti-(VCA-EBNA-EA) IgGs.

FIG. 14 illustrates the comparison of the detection of the IgGs in serafrom subjects who have been previously infected, obtained by IFA (FIG.14A) or by an ELISA test using the reagent according to the inventionVCAp18/SEQ ID No. 2+MIXO(P,G) (FIG. 14B) with the IgM reactivity of serafrom patients who have previously been infected, obtained with an ELISAtest using a reagent according to the invention VCAp18/SEQ ID No.2+MIXO(P,G).

FIG. 15 illustrates the comparison of the absorbance value obtained forthe detection of the IgGs in sera from primary-infected subjects, with aVCA-EBNA-EA Behring ELISA test or a VCAp18/SEQ ID No. 2+MIXO(P,G) ELISAtest according to the invention with the IgM reactivity obtained withthe aid of an ELISA test using a reagent according to the inventionVCAp18/SEQ ID No. 2+MIXO(P,G).

FIG. 16 illustrates an amino acid replacement matrix modified inrelation to that of H. M. GEYSEN (reference cited above) and takes intoaccount the symmetry; the values in bold characters are present in theGeysen matrix, the 0 values which are statistically not significant arekept arbitrarily or replaced by the value obtained by the symmetricalreplacement.

It should be clearly understood, however, that these examples are givensolely by way of illustration of the subject of the invention and do notconstitute in any manner a limitation thereto.

EXAMPLE 1 Preparation of the Reagents According to the Invention

a) Synthesis of the Peptide

The peptide VCAp18/SEQ ID No. 2 is synthesized using the conventionalsolid phase strategy of the Boc-benzyl (or Fmoc) type, in an automatedpeptide synthesizer (model 430A, Applied Biosystems Inc.). The groupsfor protecting the side chains are the following: Asn (Trt), Gln (Trt),Asp (OChx), Glu (OChx), Ser (Bzl), Thr (Bzl), Arg (Tos), Cys (4-MeBzl)and His (Dnp) (see R. C. SHEPPARD, Peptide, Synthesis, Comp. Org. Chem.,1979, 5, 321-363).

The amino acids are introduced using the HBTU/HOBt activation protocolwith a systematic double coupling with a resin Boc-Gln-Pam (AppliedBiosystems). After thiolysis of the dinitrophenyl group Dnp, followed bya final deprotection and a cleavage with hydrofluoric acid, the cleavedand deprotected peptide is precipitated and washed with cold diethylether and then dissolved in 5% acetic acid and freeze-dried.

The peptide is purified at more than 90% on a 100 A Nucleosil C18preparative RP-HPLC column 5 mm×250 mm (Macherey Nagel, Düren, FRG) andthe said peptide is then characterized.

The homogeneity is confirmed by analytical HPLC on a Vydac C18 columneluted with a system of solvents (TFA-acetonitrile-water), in a Shimadzuapparatus. The identity is confirmed by the determination of the aminoacid composition and by mass spectrometry recorded on a Bio Ion 20plasma mass spectrometer (Bio Ion AB, Upsala, Sweden) ([M+H]⁻ calc.:2494.7; found: 2495.4).

b) Preparation of the Different Mixotopes

These are prepared as described in H. GRAS-MASSE et al., PeptideResearch, 1992, 5, 4, 211-216.

Briefly, equimolar quantities of protected amino acids are weighed andare used in the coupling reactions.

To compensate for the differences in kinetics in the reactivities of thedifferent amino acids, a first coupling is carried out with 1 mmol(total quantity) of Boc-amino acid (or of a mixture of Boc-amino acids).A second coupling, using 2 mmol (total quantity) is then systematicallycarried out. After cleaving with hydrofluoric acid, the crude peptide isdissolved in TFA (30 ml) and precipitated by adding the said peptide toa cold diethyl ether solution (300 ml).

After centrifugation, the precipitate is dissolved in water andfreeze-dried. After air-oxidation of the solution at neutral pH, themixotopes are purified by gel filtration on a TSK HW40S column (Merck,Darmstadt, FRG). An aliquot of each purified imixotope is subjected tototal acid hydrolysis for 24 hours with a 6N HCl:phenol (10:1) mixture,for the determination of the amino acid composition.

c) Examples of Different Reagents in Accordance with the Invention

The sequence of the peptide SEQ ID No. 2 and of the mixotopes which arederived therefrom are represented in Table I below.

TABLE I Number of Antigen Sequence peptides VCAp18CAVDTGSGGGGQPHDTAPRGARKKQ 1 (SEQ ID No. 2) MIXO AVDTGSGGGGQPHDTAPRGARKKQ 16 777 216 C  GINSSASSSSNAGNSGAKSGKRRN MIXO(P) AVDTGSGGGGQ HDTA RGAKKKQ 4 194 304 C           P     P  GIESSHSSSSNGESG KSCKRRN MIXO(P,G)  AVDT      Q HDTA RGARKKQ 65 536 C     GSGGGGP    P  GIES       N GESG KSGKRRN

Reagent 1: peptide SEQ ID No. 2+mixotope MIXO mixture (completedegeneration of the structure as presented in Table I above),

Reagent 2: peptide SEQ ID No. 2+mixotope MIXO(P) mixture,

Reagent 3: peptide SEQ ID No. 2+mixotope MIXO(P,G) mixture.

These reagents are preferably attached to a solid support (microplate)at a concentration of 0.1 μg/well, for the peptide VCAp18/SEQ ID No. 2and at a concentration of 10 μg/well for the mixotopes.

EXAMPLE 2 Immunoenzymatic Test Using a Reagent According to theInvention for the Serodetection of EBV

A. Materials and Methods

ELISA

Microtitre plate wells (Nunc, Maxisorp, Rocksilde, Denmark) are coatedovernight at 4° C., either with 0.2 ml of peptide VCAp18/SEQ ID No. 2,or with a mixotope (0.5 μg/ml in 50 mM of NaHCO₃, pH 9.6), orsequentially with 0.2 ml of peptide VCAp18/SEQ ID No. 2 (0.5 μg/ml) and0.2 ml of mixotope (50 μg/ml).

Each well is then washed with a 0.01 M phosphate buffer comprising 1.8%NaCl, pH 7.4 (PBS) and the binding sites in excess are blocked withalbumin (addition of 0.3 ml of 2% BSA (bovine serum albumin) in PBS, at37° C., for 60 minutes).

After 3 washes with 0.3 ml of PBS 0.5%-Tween 20 (Sigma) (PBS-T buffer),the human sera to be tested are diluted 1/50 in PBS-T comprising 2%bovine serum albumin (BSA) and are incubated in wells containing thereagent according to the invention as specified above, for 120 minutesat 37° C., in a humidified atmosphere.

After 4 washes, the peroxidase-goat antibody anti-human IgG-A-Mconjugates (diagnostic Pasteur), diluted 1/10,000 in PBS-T buffercomprising 2% BSA, are incubated for 60 minutes at 37° C.

The conjugated antibody, which binds to the Ig's attached to thesupport, is revealed for its peroxidase activity, using as substrateo-phenylenediamine dihydrochloride and H₂O₂, in a 0.05 M citrate buffer,pH 5.5, for 30 minutes in the dark and at room temperature.

The reaction is blocked by the addition of 4N H₂SO₄ (50 μl). Theabsorbance is recorded against a blank at 492 nm (A₄₉₂), with amultichannel automated reader (M_(R) 5000, Dynatech).

The mean A₄₉₂+3 standard deviations (SD) of the EBV-negative samples isused as cut-off value in the ELISA tests.

Measurement of the Binding Affinity to the Antibody

The specificity of the binding of the positive sera to the differentmixotopes in solid phase is evaluated by the absorption of theantibodies by the native antigen VCAp18/SEQ ID No. 2 in solution, usingthe method of B. FRIGUET et al. (J. Immunol. Methods, 1985, 77,305-319).

This method is based on the measurement of the free antibodyconcentration by an indirect ELISA method when the antigen VCAp18/SEQ IDNo. 2 and the antibodies are in equilibrium in solution.

The antigen VCAp18/SEQ ID No. 2, at different concentrations (10⁻¹⁰ M to2×10⁻⁶ M), is first incubated in solution (PBS-T buffer+2% BSA) with anEBV-positive serum, at a constant concentration (1/50) until theequilibrium state is reached.

After incubating for 18 hours at 4° C., 200 μl of each mixture istransferred and incubated for 60 min at 20° C. in the wells of amicrotitre plate, previously coated with peptide VCAp18/SEQ ID No. 2(0.2 ml, corresponding to 0.5 μg/ml) or a mixotope (50 μ/ml, 0.2 ml), in50 mM NaHCO₃, pH 9.6.

After washing with PBS-T buffer, the bound immunoglobulins are detectedby addition of peroxidase-coupled goat antibodies anti-human IgG-A-Ms.

The conjugated antibody, which binds to the Ig's, is revealed by theperoxidase activity as described above. This method gives thedisplacement curves for the A/Ao bond relative to log(ao). A preciseestimation of the mean affinity of the serum containing the anti-VCAp18antibodies is given by the equation Ao/(Ao−A)=1/ν=1+Kd/ao, in which aois the total soluble antigen concentration, A and Ao are the absorbancevalues at 492 nm with or without blocking antigen, respectively, and νis the bound antibody fraction, if the different conditions set out inFRIGUET et al. are satisfied.

B. Results

a) Binding Serum Antibodies—C-terminal Domain of the Peptide VCAp18/SEQID No. 2 (ELISA VCAp18/SEQ ID No. 2)

The reactivity of the anti-VCA human IgG-M-As with respect to thepeptide VCAp18/171-194 is analysed by an ELISA test, on different sera:46 EBV-positive sera, and 28 EBV-negative sera, selected after carryingout a Behring ELISA test and confirmed by IFA (detection of the anti-VCAIgGs, Immunoconcept, USA).

As specified in W. M. J. Van GRUNSVEN, J. Infect. Dis., 1994, 170,13-19, the C-terminal domain of the VCAp18 protein shows a highimmunoreactivity (FIG. 1A).

When it coats microtitre plates at a concentration of 0.1 μg/well, thispeptide VCAp18/SEQ ID No. 2 is recognized by the antibodies of most ofthe EBV-positive sera.

Using 3 standard deviations above the mean value obtained with thecontrol negative sera as lower limit of detection, a high bindingsensitivity (89%) and an optimal specificity (100%) are observed in theanalysis of the anti-VCAp18 response (FIG. 1A).

To evaluate the possibility of increasing the sensitivity of the test,as a function of the quantity of peptides covering the plates, anotherconcentration was tested for the coating (quantity coating the plates)namely 1 μg/wells; in such a case, a decrease in specificity to 89% isobserved (FIG. 1C).

Comparison of the IF titres and of the absorbance values obtained inELISA, for each serum, does not show any clear correlation, essentiallybecause of a high variability of the absorbance values obtained in ELISAfor the sera having intermediate or low IF titres (FIG. 1B).

The EBV-positive sera, which escape detection by ELISA ( in FIG. 1B),show IF titres which are detectable at the 160^(th) or at the 320^(th)(limiting dilutions), confirming that the VCA antigen is effectivelyrecognized by these four peptide VCAp18-negative sera.

b) Binding Serum Antibodies Mixotopes (ELISA VCA Mixotope)

The mixotopes were tested as antigens in solid phase, at twoconcentrations, namely 0.1 μg and 10 μg/well (FIG. 2).

After coating the wells at a concentration of 0.1 μg, the sensitivity isclearly insufficient; only 26, 10 and 10 of the 46 IgG-A-M-positive serareact with MIXO, MIXO(P) and MIXO(P,G) respectively (FIGS. 2A, C, E).

After coating with the mixotopes at a concentration of 10 μg/well, thedetection of the positive sera is much more sensitive with an increasein the absorbance values. 46, 36 and 27 of the 46 EBV-positive sera aredetected with the mixotopes MIXO, MIXO(P) and MIXO(P,G) respectively(FIGS. 2B, D, F). However, the cut-off value is also higher, because ofan increased variation in the absorbance values observed with thenegative control sera.

Surprisingly, the loss of binding specificity (11 false-positive sera),is observed only when the antigen MIXO is used (FIGS. 2B, D, F), nofalse-positive is observed with the other two mixotopes.

Comparison of the IF titres and of the absorbance values obtained inELISA for each of the 46 IFA-positive sera are illustrated in FIG. 3.The absorbance values observed in ELISA are increased relative to theresults obtained with the IF test; these results are particularlyadvantageous for the sera having low or intermediate IFA titres.

The VCAp18-negative IF-positive tests are symbolized by black squares inFIG. 3.

c) Serum Antibodies-reagent Binding According to the Invention(Combinations Peptide VCAp18/SEQ ID No. 2+Mixotope) (ELISA VCAp18/SEQ IDNo. 2+Mixotope)

To show the increase in the sensitivity of detection with the aid of thereagent according to the invention, combinations are used which comprisethe peptide VCAp18/SEQ ID No. 2 coating a microtitre plate in an amountof 0.1 μg/well with various mixotopes covering the microtitre plate inan amount of 10 μg/well.

As illustrated in FIGS. 4(A, B, C), all the EBV-positive sera aredetected with the three combinations.

A heterogeneity in the results, with the ELISA test, is observed withthe EBV-negative sera when the degeneration of the mixotope increases.As illustrated in FIGS. 4A and 4B, the combinations MIXO and MIXO(P)with the peptide VCAp18/SEQ ID No. 2 result in the presence of 4 or 5false-positive sera. However, the combination with the antigen MIXO(P,G)provides an optimal binding sensitivity (100%) and an optimalspecificity (100%) in the VCA-EBV immunoenzymatic serodiagnosis (FIG.4C).

The results obtained are illustrated in Table II below.

TABLE II Antigen Coating Detection solid condition Ig Serum EBV NegativeSpecificity Serum EBV Positive Sensitivity phase (μg/well) isotype N + −% N + − % SEQ ID 0.1 Ig (G-A-M) 28 0 28 100 46 42 4 91 No. 2 1 Ig(G-A-M) 28 3 25  89 46 44 2 95 MIXO 0.1 Ig (G-A-M) 28 2 26  92 46 26 20 56 10 Ig (G-A-M) 28 11  17  60 46 44 2 95 MIXO (P) 0.1 Ig (G-A-M) 28 028 100 46 10 36  21 10 Ig (G-A-M) 28 0 28 100 46 36 10  78 MIXO (P,G)0.1 Ig (G-A-M) 28 0 28 100 46 10 36  21 10 Ig (G-A-M) 28 0 28 100 46 2722  52 SEQ ID 0.1 + 10 Ig (G-A-M) 28 3 25  89 46 46 0 100  No. 2 + MIXOSEQ ID 0.1 + 10 Ig (G-A-M) 28 4 24  85 46 46 0 100  No. 2 + MIXO (P) SEQID 0.1 + 10 Ig (G-A-M) 28 0 28 100 46 46 0 100  No. 2 + MIXO (P,G)

d) Relevance of the Combinations of Peptide VCAp18/Mixotopes for theSerodiagnosis of EBV

A more detailed analysis of the distribution of the individual responsesto the peptide VCAp18/SEQ ID No. 2, to its mixotopes and to theircombinations (reagent according to the invention) is illustrated in FIG.5.

All the sera exhibit an absorbance value of less than 1.3 with an ELISAtest using only the peptide VCAp18/SEQ ID No. 2 were selected. Eachserum is represented by 4 symbols corresponding to the absorbance valuesobtained when they are reacted with the different antigens in solidphase.

In some cases, the signal is clearly increased when the mixotopes areused as antigens in solid phase including for the IF-positive-VCAp18/SEQID No. 2-negative sera.

As illustrated in FIG. 5C, almost all the absorbance values obtainedwhen the positive sera are tested with the reagent according to theinvention are greater than the absorbance values obtained with thepeptide VCAp18/SEQ ID No. 2 alone.

The 3 combinations efficiently detect all the EBV-positive sera, whichexhibit a low signal with the mixotopes.

The signal obtained with the sera which are negative with thecombinations according to the invention is also generally lower thanwhen these sera are tested with the peptide VCAp18/SEQ ID No. 2 alone,except when the completely degenerate construct MIXO is used.

However, the completely degenerate mixotopes obtained from peptidesderived from the C-terminal portion of the VCAp18 protein and containingno proline, for example, effectively confer the desired properties onthe reagent according to the present invention.

e) Evaluation of the Avidity of the Mixotopes for the Serum AntibodiesCompared with the Peptide VCAp18/SEQ ID No. 2, by ELISA

The peptide VCAp18/SEQ ID No. 2 is used in the ELISA inhibitionexperiments to evaluate its capacity to inhibit the binding of theEBV-positive human sera to the different mixotopes, used in solid phase.

FIG. 6 represents a few inhibition trials.

As illustrated in this FIG. 6, the binding of the human sera to the 3mixotopes may be specifically and strongly inhibited by increasing theconcentrations of peptide VCAp18/SEQ ID No. 2.

In addition, the peptide VCAp18/SEQ ID No. 2 in solid phase and thedifferent mixotopes in solid phase give parallel displacement curves,indicating the presence of a common population of antibodies which issensitive to the native peptide and to the degenerate peptides.

FIG. 7 represents the Klotz plots for binding to the antibodies of theEBV-positive sera, measured in ELISA by the method of FRIGUET et al.,cited above. The values of the dissociation constants are deduced fromthe linear regression of the results, expressed in FIG. 6. An increaseby a factor of 10 to 400 in the affinity constants is observed for thedifferent mixotopes, compared with the peptide VCAp18/SEQ ID No. 2.

The mean Kd value for VCAp18/171-194, MIXO, MIXO(P) and MIXO(P,G) inthese experiments is respectively 0.81±0.62 mM, 0.06±0.01 mM, 2.40±1.54nM and 4.75±3.75 nM.

EXAMPLE 3 Immunoenzymatic Test Using a Reagent According to theInvention for the Differential Serodetection of the Anti-VCA Human IgGs,IgMs and IgAs and its Role in the Diagnosis of Different Pathologies

a) Binding Serum Antibodies Reagent VCAp18/SEQ ID No. 2+MIXO(P,G)According to the Invention in Healthy Carriers

The reactivity of the anti-VCA human IgG-A-Ms, IgGs, IgAs and IgMs withrespect to the reagent according to the invention VCAp18/SEQ ID No.2+MIXO(P,G), is analysed with the aid of an ELISA test, using the seracharacterized in IF as EBV-negative and EBV-positive (46 EBV-positivesand 28 EBV-negatives).

The cut-off is defined as three standard variations above the mean valuefor the EBV-negative sera, determined for each isotype.

With this criterion, no false-positive serum is observed in any ELISA.

As illustrated in Example 1, c) above (see also FIG. 4C), the reagentpeptide VCAp18/SEQ ID No. 2+MIXO(P,G), used as antigen in solid phase,shows an optimal binding sensitivity of 100% and a specificity of 100%in the IgG-A-M serological diagnoses of EBV by an immunoenzymaticmethod.

The same set of sera which are positive by IF was tested for its IgM,IgG and IgA reactivity, with an ELISA test using as reagent a VCAp18/SEQID No. 2+MIXO(P,G) mixture.

To carry out the isotype-specific tests, the procedure is carried out asfollows:

The procedure is carried out as specified in Example 1, for thepreparation of the microtitre plates.

However, before the incubation of the sera to be analysed, under theconditions specified in Example 1, the sera used for the analysis of theIgMs may be treated:

to remove the IgGs (preadsorption of the IgGs or pretreatment with ananti-human IgG serum (Diagnostic Pasteur, Marnes-La-Vallée, France) or

in order to resolubilize the IgMs with a solution absorbing therheumatoid factor (Behringwerke AG, Marburg, Germany),

in accordance with the manufacturers' instructions.

For the revealing, the procedure is carried out as follows:

After 4 washes, the conjugates of goat antibodies (also called secondantibodies) directed against the human Ig(G-A-M)s, the IgGs, the IgAsand the IgMs (Diagnostic Pasteur, Marnes-La-Coquette, France), diluted1/10,000 in a PBS-T+2% BSA buffer are incubated for 60 min at 37° C.

The second conjugated antibodies which bind to the serum Ig's attachedto the support are revealed as specified in Example 1.

As illustrated in FIG. 8B, all the sera with the exception of 2 reactpositively with the IgGs; however, these two IgG-negative sera have highIgA and IgM reactivities, as indicated by the black squares in FIG. 8B.These results are in agreement with the 100% binding sensitivity and thespecificity obtained in the IgG-A-M test (FIG. 4C or FIG. 8A) and withthe fact that the sera from healthy carriers (infected in the past)exhibit a low IgA and IgM reactivity (FIG. 8B).

However, 91% of these sera from subjects who were infected in the past(healthy carriers) are IgM-positive, among which 78% have residuallevels of IgM (OD less than 1), compared with the absorbance valueobtained for the IgGs; only 39% of the said sera are IgA-negative; thisis a surprising result since most of the patients were considered ashealthy carriers or as convalescents, that is to say IgM andIgA-negative.

Only one serum was detected as having high levels of IgM and of IgG,whereas it was IgA-negative, probably indicating a reinfected orprimary-infected patient.

To show the specificity of the IgM response, the sera from the healthycarriers were treated with IgG-precipitating reagents: serum treatedwith an anti-IgG serum or with IgM-resolubilizing reagents or reagentsallowing decomplexing of the IgMs: serum treated with a solution capableof absorbing the rheumatoid factor.

FIG. 9 compares the three ELISAs carried out (untreated serum, serumtreated with absorbent and serum treated with anti-IgG serum). Nosignificant difference is detected, a low signal being observed for theELISA carried out on sera treated with the reagents precipitating theIgGs.

The VCAp18/SEQ ID No. 2+MIXO(P,G) ELISA test for the detection of theIg(G-A-M)s or of the IgGs were compared in FIG. 10 with a conventionalimmunofluorescence method using as antigen VCA or a combinationVCA-EBNA-EA for the detection of the IgGs in each of the 46 IFA-positivesera (Behring method).

As illustrated in FIGS. 10A and 10C, to high IFA titres correspond highabsorbance values with the VCAp18/SEQ ID No. 2+MIXO(P,G) ELISA tests forthe detection of the Ig(G-A-M)s or of the IgGs.

Moreover, a high increase in the values obtained with the VCAp18/SEQ IDNo. 2−MIXO(P,G) ELISA test is observed with the intermediate and low IFtitres.

This situation is particularly important for the detection of theIgG-A-Ms and demonstrates the relevance of the IgG-A-M detection (FIGS.10A, C).

The same type of result is observed in FIG. 10D which compares an ELISAtest using the VCAp18/SEQ ID No. 2+MIXO(P,G) reagent and a commercialBehring ELISA, for the detection of the IgGs.

The two EBV-positive sera, which escape detection with the aid of thereagent VCAp18/SEQ ID No. 2−MIXO(P,G), exhibit low or intermediate IFtitres (80^(th) and 320^(th)) and are below the mean value observed inthe Behring ELISA test for the detection of the IgGs.

This result is not inconsistent since another set of antigens includingEA and EBNA is used in the Behring test.

FIG. 10B compares the VCAp18/SEQ ID No. 2+MIXO(P,G) ELISA test, for thedetection of the IgG-A-Ms according to the invention, and the BehringELISA test, for the detection of the IgGs. No clear correlation isobserved between the IgG and IgG-A-M reactivities, suggesting that thecombination according to the invention is very specific and contributesto the binding of all the human isotypes, including type M considered asthe isotype with the lowest affinity and which is produced right at thebeginning of the infection.

b) Study of the Binding of the Serum Antibodies to the Reagent Accordingto the Invention VCAp18/SEQ ID No. 2+MIXO(P,G) in Primary-infectedPatients

The reactivity of the anti-VCA human IgG-A-Ms, IgGs, IgAs and IgMs withrespect to the reagent according to the invention VCAp18/SEQ ID No.2+MIXO(P,G) is analysed with the aid of an ELISA test, using the 28EBV-negative sera selected as controls and the 40 EBV-positive seraobtained from primary-infected patients and giving positive results withan ELISA test (EBNA-VCA-EA) for the detection of the IgMs and negativeresults with a Behring ELISA test for the detection of the anti-EBNA1human IgGs.

The results obtained with the ELISA test using the reagent according tothe invention VCAp18/SEQ ID No. 2+MIXO(P,G) for the detection of theIgG-A-Ms are presented in FIG. 11A and a detailed study of the isotyperesponse of the different sera is illustrated in FIG. 11B.

Only six EBNA1-negative sera give a result above the cut-off as regardsthe reactivity with respect to the IgG-A-Ms (FIG. 11A).

All these sera belong to the group consisting of the IgG-negative sera(40%). Two of them do not react with any test and are considered asfalse-negatives.

Furthermore, they are the only two to be IgM-negative (FIG. 11B).Following this IgM-negativity, these two sera were tested inimmunofluorescence (IF) for confirmation of their seropositivity. Thetitres obtained correspond to values at one-tenth and one-fortieth.These very low titres indicate great precociousness of the EBVinfection. One explanation might be the detection of another antigensuch as EA included in commercial kits or more probably a cross-reactionwith another virus of the Herpes group (CMV, HSV and the like).

Moreover, the other 4 sera which are also IgG-negative have low IgM andIgA levels indicating an early phase of primary infection.

The specificity of the IgM reactivity was studied by neutralizing theIgG reactivity with precipitating agents.

As illustrated in FIGS. 12A and 12C, the untreated sera and the treatedsera (RF-absorbent) do not exhibit any difference in reactivity.

On the other hand, the preadsorption of the IgGs induces twofalse-negative results (FIG. 12B). However, the latter two sera areIgG-A-M-positive. One of them proved only IgA-positive, whereas theother provides a high IgG response and a low IgA response.

FIG. 13 compares an ELISA test using a reagent according to theinvention (VCAp18/SEQ ID No. 2+MIXO(P,G) for the detection of the IgGsand a Behring ELISA test for the specific detection of the anti-EBVIgMs, in which a nuclear extract of the cells infected with an EBV isused as antigen.

No correlation is observed between the values obtained with these twoELISA tests. A shift in the absorbance values is observed with thereagent according to the invention. A serum giving a result which isdifficult to interpret with the Behring test is efficiently detectedwith the ELISAs, using a reagent according to the invention, for thespecific detection of the anti-VCA IgMs and IgGs.

c) Relevance of the Reagent According to the Invention VCAp18/SEQ ID No.2+MIXO(P,G) for the Differential Serodiagnosis of the Isotypes ProducedDuring an EBV Infection

Discrimination between a previous infection (healthy carrier) and aprimary infection was up until now based on the IgM and IgG reactivityobtained with the Behring ELISA tests using a VCA+EBNA+EA reagent forthe detection of the IgMs and IgGs.

FIGS. 14 and 15 illustrate the comparative results obtained with thereagent according to the invention VCAp18/SEQ ID No. 2+MIXO(P,G) for thedetection of the human IgM response, in two EBV-positive populationsstudied with reference to their IgG level determined by the methodaccording to the present invention and by conventional methods.

The absorbance values obtained with patients who have previously had aninfection (healthy carriers) are distributed along the x-axis (FIG.14A). This situation is in agreement with the results obtained inconventional serology with these type of patients (IgM-negative andIgG-positive, as determined with an immunofluorescence test).

Only one of these sera gave a strong IgM-positive signal with thereagent according to the invention VCAp18/SEQ ID No. 2+MIXO(P,G),indicating a primary infection (FIG. 14A); in addition, a strong IgGsignal is obtained whereas by immunofluorescence only a weak IgG signalis observed.

This result confirms a reinfection (or a convalescence phase) since theIgA response is negative; this result shows the importance of thereagent according to the invention in the detection of the differentisotypes.

A shift to the right, along the x-axis is in addition observed in theELISA test carried out with the reagent according to the invention.

In the case of primary-infected patients, the values obtained with theELISA tests are distributed along the y-axis.

In fact FIGS. 15(A and B) show a diagonal distribution.

The M and G isotypes appear sequentially, but almost simultaneously,after the onset of the infection.

Consequently, it is not surprising to detect the G isotype in the serafrom primary-infected patients.

The relatively large subset of sera not situated along the diagonal oralong the y-axis, but near the x-axis indicates a latent or persistentphase of the infection for these sera since most of them areIgA-positive. This situation appears less in FIG. 15B in which areduction is observed in the number of sera, along the x-axis.

Surprisingly, the reagents according to the invention allow both thedetection of the overall Ig reactivity (IgG-A-M) and the detection ofthe reactivity of each isotype (IgG, M or IgA).

The results described above are illustrated in Table III below.

TABLE III Antigen Coating Detection solid condition Ig Serum EBVNegative Specificity EBV Positive Sensitivity phase (μg/well) isotypeN + − % Serum N + − % VCAp18/ 0.1 Ig(G-A-M) 28 0 28 100 L 46 42  4 91SEQ ID P 40 24 16 60 No. 2 1 Ig(G-A-M) 28 6 22  78 L 46 44  2 95 P 40 34 6 85 VCAp18/ 0.1 IgM 28 0 28 100 L 46 nd nd SEQ ID P 40 29 11 72 No. 21 IgM 28 0 28 100 L 46 nd nd P 40 38  2 95 VCAp18/ 0.1 IgA 28 0 28 100 L46 nd nd SEQ ID P 40 25 15 62 No. 2 1 IgA 28 0 28 100 L 46 nd nd P 40 2911 72 VCAp18/ 0.1 IgG 28 0 28 100 L 46 nd nd SEQ ID P 40 13 27 32 No. 21 IgG 28 1 27  96 L 46 nd nd P 40 24 16 60 VCAp18/ 0.1 + 10 Ig(G-A-M) 280 28 100 L 46 46  0 100* SEQ ID P 40 34  6 85 No. 2 +   1 + 10 Ig(G-A-M)28 0 28 100 L 46 nd nd MIXO (P,G) P 40 39  1  97* 0.1 + 10 IgM 28 0 28100 L 46 42  4 91 P 40 38  2 95   1 + 10 IgM 28 0 28 100 P 40 38  2 950.1 + 10 IgA 28 0 28 100 L 46 28 18 61 P 40 30 10 75   1 + 10 IgA 28 028 100 P 40 33  7 83 0.1 + 10 IgG 28 0 28 100 L 46 44  2 96 P 40 24 1660   1 + 10 IgG 28 0 28 100 L 46 nd nd P 40 29 11 72

This table shows the results obtained for 86 sera from subjects who arehealthy carriers or from primary-infected subjects when the peptideVCAp18/SEQ ID No. 2 is used alone; 60% and 51% of positive responses areobserved for these two populations, respectively, without loss ofspecificity.

Legend to Table III: nd=not determined; L=sera from healthy carriers(IgM-negative and IgG-positive); P=sera from primary-infected patients(IgM-positive and IgG in most cases). There is no indication as regardsthe IgAs with the conventional ELISAs.

When a reagent according to the invention VCAp18/SEQ ID No. 2+MIXO(P,G)is used, the percentages of positive results obtained in the twopopulations reach 97% and 100%, respectively. The latter result (TableIII, *) depends on the concentrations for use which are selected for thepeptide VCAp18/SEQ ID No. 2 and relates to the overall IgG-A-Mreactivity.

If all the results are observed, a good correlation (98.8%) is observedbetween the conventional methods and the method using a reagentaccording to the invention (good sensitivity).

This demonstrates the relevance of the IgG-A-M serodetection, which isusually considered not to be very sensitive and to be only IgG-specific.

In fact, with the reagents according to the present invention, when theindividual IgG, IgA and IgM sensitivities are compared with the resultsobtained with the IgG-A-M detection according to the invention, thelatter corresponds approximately to the sum of the results obtained foreach isotype; in particular, none of the isotype-positive sera wasnegative, when an overall Ig(GAM) reactivity is detected according tothe method according to the present invention.

These results show the specificity and the sensitivity of the reagentaccording to the invention.

In addition, these results show that the sera from subjects who havepreviously had an infection have residual levels of IgM.

The IgM reactivities are surprising because they are in contrast to theresults obtained with the prior art tests.

These results suggest in particular that the IgMs persist with the IgGsfor a long period after infection.

As evident from the above, the invention is not at all limited to itsembodiments, implementations and applications which have just beendescribed more explicitly; it embraces on the contrary all the variantswhich may occur to the specialist in this field, without departing fromthe framework or the scope of the present invention.

7 1 176 PRT Epstein-Barr virus 1 Met Ala Arg Arg Leu Pro Lys Pro Thr LeuGln Gly Arg Leu Glu Ala 1 5 10 15 Asp Phe Pro Asp Ser Pro Leu Leu ProLys Phe Gln Glu Leu Asn Gln 20 25 30 Asn Asn Leu Pro Asn Asp Val Phe ArgGlu Ala Gln Arg Ser Tyr Leu 35 40 45 Val Phe Leu Thr Ser Gln Phe Cys TyrGlu Glu Tyr Val Gln Arg Thr 50 55 60 Phe Gly Val Pro Arg Arg Gln Arg AlaIle Asp Lys Arg Gln Arg Ala 65 70 75 80 Ser Val Ala Gly Ala Gly Ala HisAla His Leu Gly Gly Ser Ser Ala 85 90 95 Thr Pro Val Gln Gln Ala Gln AlaAla Ala Ser Ala Gly Thr Gly Ala 100 105 110 Leu Ala Ser Ser Ala Pro SerThr Ala Val Ala Gln Ser Ala Thr Pro 115 120 125 Ser Val Ser Ser Ser IleSer Ser Leu Arg Ala Ala Thr Ser Gly Ala 130 135 140 Thr Ala Ala Ala SerAla Ala Ala Ala Val Asp Thr Gly Ser Gly Gly 145 150 155 160 Gly Gly GlnPro His Asp Thr Ala Pro Arg Gly Ala Arg Lys Lys Gln 165 170 175 2 71 PRTArtificial Sequence Synthetic Peptide 2 Xaa Ala Leu Ala Val Ala Leu AlaSer Pro Thr His Arg Gly Leu Tyr 1 5 10 15 Ser Glu Arg Gly Leu Tyr GlyLeu Tyr Gly Leu Tyr Gly Leu Tyr Gly 20 25 30 Leu Asn Pro Arg His Ile SerAla Ser Pro Thr His Arg Ala Leu Ala 35 40 45 Pro Arg Ala Arg Gly Gly LeuTyr Ala Leu Ala Ala Arg Gly Leu Tyr 50 55 60 Ser Leu Tyr Ser Gly Leu Asn65 70 3 30 PRT Artificial Sequence Synthetic Peptide 3 Ser Thr Ala ValAla Gln Ser Ala Thr Pro Ser Val Ser Ser Ser Ile 1 5 10 15 Ser Ser LeuArg Ala Ala Thr Ser Gly Ala Thr Ala Ala Ala 20 25 30 4 58 PRT ArtificialSequence Synthetic Peptide 4 Ser Thr Ala Val Ala Gln Ser Ala Thr Pro SerVal Ser Ser Ser Ile 1 5 10 15 Ser Ser Leu Arg Ala Ala Thr Ser Gly AlaThr Ala Ala Ala Ser Ala 20 25 30 Ala Ala Ala Val Asp Thr Gly Ser Gly GlyGly Gly Gln Pro His Asp 35 40 45 Thr Ala Pro Arg Gly Ala Arg Lys Lys Gln50 55 5 30 PRT Artificial Sequence Synthetic Peptide 5 Ser Val Ser SerSer Ile Ser Ser Leu Arg Ala Ala Thr Ser Gly Ala 1 5 10 15 Thr Ala AlaAla Ser Ala Ala Ala Ala Val Asp Thr Gly Ser 20 25 30 6 6 PRT ArtificialSequence Synthetic Peptide 6 Gly Ser Gly Gly Gly Gly 1 5 7 8 PRTArtificial Sequence Synthetic Peptide 7 Ala Ala Ala Ser Ala Ala Ala Ala1 5

What is claimed is:
 1. A reagent for diagnosing a viral infection,comprising (1) an immunodominant fragment of a protein of the viruscomprising at most 60 amino acids; and (2) a mixture of convergentcombinatory peptides obtained by systematic or partial replacement ofeach amino acid of the immunodominant fragment with another amino acid,according to a suitable replaceability matrix.
 2. The reagent accordingto claim 1, wherein the viral infection is Epstein Barr viral infectionand the reagent comprises (1) a C-terminal fragment of at most 60 aminoacids of SEQ ID NO:1; and (2) a mixture of convergent combinatorypeptides derived from the C-terminal fragment of SEQ ID NO:1.
 3. Thereagent according to claim 2, wherein the C-terminal fragment isselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, and SEQ ID NO:5.
 4. The reagent according to claim 2, wherein thecombinatory peptides are obtained by systematic replacement of eachamino acid of the C-terminal fragment with another amino acid.
 5. Thereagent according to claim 2, wherein the combinatory peptides areobtained by partial replacement of each amino acid of the C-terminalfragment with another amino acid.
 6. The reagent according to claim 5,wherein the C-terminal fragment peptide is SEQ ID NO:2 and at least oneof the proline residues, and the sequence Gly Ser Gly Gly Gly Gly (SEQID No.6) are conserved.
 7. The reagent according to claim 5, wherein theC-terminal fragment peptide is SEQ ID NO:3, and at least one of theproline residues, the serine residues, and the alanine residues of therepetitive sequences are conserved.
 8. The reagent according to claim 5,wherein the C-terminal fragment is SEQ ID NO:4, and at least one of theserine residues, the proline residues, the alanine residues, thesequence Gly Ser Gly Gly Gly Gly (SEQ ID No. 6), and the sequence AlaAla Ala Ser Ala Ala Ala Ala (SEQ ID No. 7) of SEQ ID NO:4 are conserved.9. The reagent according to claim 5, wherein the C-terminal fragment isSEQ ID NO:5, and at least one of the serine residues, the alanineresidues, and the sequence Ala Ala Ala Ser Ala Ala Ala Ala (SEQ ID No.7) of SEQ ID NO:5 are conserved.
 10. The reagent according to claim 1,wherein the reagent is attached to a solid support.
 11. The reagentaccording to claim 2, wherein the ratio of the C-terminal fragment ofSEQ ID NO:1 to the mixture of convergent combinatory peptides if from1:10 to 1:100.
 12. Method for the diagnosis of a viral infection, by animmunoenzymatic method, characterized in that it uses a diagnosticreagent according claim
 1. 13. Diagnostic method, characterized in that,for the diagnosis of an EBV infection by an immunoenzymatic method, itcomprises: bringing a serum to be analysed into contact with a reagentaccording to claim 2, the addition of anti-human Ig antibodies coupledto an enzyme, and the qualitative and/or quantitative revealing of theanti-VCA antibodies which may be present in the serum to be analysed byaddition of the enzyme substrate.
 14. Method according to claim 13,characterized in that it comprises: the attachment of a reagentaccording to any one of claims 2 to 11 onto a support, such as amicrotitre plate, the addition of the serum to be analysed, and thedetection of the attachment of the anti-VCA antibodies present in thesaid serum by addition of anti-human Ig (G-A-M) antibodies coupled to anenzyme, and the qualitative and/or quantitative revealing in aspectrophotometer by addition of the enzyme substrate.
 15. A kit or boxfor the diagnosis of a viral infection, which comprises at least onediagnostic reagent according to claim
 1. 16. The reagent according toclaim 1, wherein the immunodominant fragment of the viral proteincomprises from 20 to 30 amino acids.
 17. The reagent according to claim2, wherein the immunodominant fragment of the viral protein comprisesfrom 20 to 30 amino acids.
 18. The reagent according to claim 10,wherein the solid support is a microtitre plate.